1. Field of the Invention
This invention relates to methods and reagents for analyzing nucleotide sequences of nucleic acids and, more particularly, to methods for analyzing nucleotide sequences wherein cross-hybridization reactions are controlled.
Determining the nucleotide sequences and expression levels of nucleic acids (DNA and RNA) is critical to understanding the function and control of genes and their relationship, for example, to disease discovery and disease management. Analysis of genetic information plays a crucial role in biological experimentation. This has become especially true with regard to studies directed at understanding the fundamental genetic and environmental factors associated with disease and the effects of potential therapeutic agents on the cell. Such a determination permits the early detection of infectious organisms such as bacteria, viruses, etc.; genetic diseases such as sickle cell anemia; and various cancers. This paradigm shift has lead to an increasing need within the life science industries for more sensitive, more accurate and higher-throughput technologies for performing analysis on genetic material obtained from a variety of biological sources.
Unique or misexpressed nucleotide sequences in a polynucleotide can be detected by hybridization with a nucleotide multimer, or oligonucleotide, probe. Hybridization is based on complementary base pairing. When complementary single stranded nucleic acids are incubated together, the complementary base sequences pair to form double stranded hybrid molecules. These techniques rely upon the inherent ability of nucleic acids to form duplexes via hydrogen bonding according to Watson-Crick base-pairing rules. The ability of single stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA) to form a hydrogen bonded structure with a complementary nucleic acid sequence has been employed as an analytical tool in molecular biology research. The oligonucleotide probe employed in the detection is selected with a nucleotide sequence complementary, usually exactly complementary, to the nucleotide sequence in the target nucleic acid. Following hybridization of the probe with the target nucleic acid, any oligonucleotide probe/nucleic acid hybrids that have formed are typically separated from unhybridized probe. The amount of oligonucleotide probe in either of the two separated media is then tested to provide a qualitative or quantitative measurement of the amount of target nucleic acid originally present.
One method for detecting specific nucleic acid sequences generally involves immobilization of nucleic acid on a solid support such as nitrocellulose paper, cellulose paper, diazotized paper, or a nylon membrane. After the target nucleic acid is fixed on the support, the support is contacted with a suitably labeled nucleic acid for about two to forty-eight hours. After the above time period, the solid support is washed several times at a controlled temperature to remove unhybridized probe. The support is then dried and the hybridized material is detected by autoradiography or by spectrometric methods.
Another method for detecting specific nucleic acid sequences employs hybridization to surface-bound arrays of sample nucleic acid sequences or oligonucleotide probes. Such techniques are useful for analyzing the nucleotide sequence of target nucleic acids. In theory, and to some extent in practice, hybridization to surface-bound arrays can provide a relatively large amount of information in a single experiment. For example, array technology has identified single nucleotide polymorphisms within relatively long (1,000 residues or bases) sequences (Kozal, M., et al., Nature Med. 7:753-759, July 1996). In addition, array technology is useful for some types of gene expression analysis, relying upon a comparative analysis of complex mixtures of mRNA target sequences (Lockart, D., et al., (1996) Nat. Biotech. 14, 1675-1680).
In many assays there may be one or more non-target nucleic acids present that have a nucleotide sequence closely related to that of the target sequence differing by only a few, e.g., one to five nucleotides. In such cases the non-target polynucleotide may then interfere with the assay by hybridizing with at least some of the target probe to produce false qualitative or quantitative results. This problem is particularly acute where the probe sequence is selected to permit assaying of various genes within a multigene family, each member of which contains a sequence closely related to the target nucleotide sequence. In analysis by array technology there is the concern that cross-hybridization may occur, which would result in false positive signals.
Approaches have been suggested for alleviating some of the above concerns. One technique involves placing on an array intentionally mismatched control probes as well as the actual probe of interest. A mismatched probe has one or more base substitutions. By observing the signal for the original probe versus the mismatched probes one can gauge specificity and perhaps even correct for cross-hybridization by subtracting some fraction of the mismatch probe signal from the signal generated by the probe of interest. In a particular approach probes are generated by constructing all possible one base substitutions at a specific position near the center of the probe and synthesizing them next to the probe of interest. However, this mismatch strategy is relatively arbitrary and multiplies by 5 the number of array locations required to evaluate the performance of a single probe. In some arrays, the percentage of array locations devoted to mismatch probes is decreased by choosing a single base substitution. However, this choice is even more arbitrary than synthesizing all possibilities at a single position.
2. Description of the Related Art
An assay for polynucleotides employing oligonucleotides to eliminate undesirable cross-reactions is discussed in U.S. Pat. No. 5,434,047 (Arnold).
Arrays of nucleic acid probes on biological chips are disclosed in U.S. Pat. No. 5,837,832 (Chee, et al.).
Sapolsky, et al., discusses polymorphism detection in U.S. Pat. No. 5,858,659.
Genome-wide expression monitoring in Saccharomyces cerevisiae is described by Wodicka, et al., in Nature Biotechnology (1997) 15:1359-1367.
Mitsuhashi, et al., in U.S. Pat. No. 5,556,749 discuss a computerized method for designing optimal DNA probes and an oligonucleotide probe design station.